Polarized epithelial cells are important in regulating the environment between external and internal compartments in an organism. Many malignant and nonmalignant diseases are characterized by changes in cell polarity. It is not known whether these are a cause or effect of the specific disease, but an understanding of the biogenesis, transport, assembly and maintenance of polarized cells may lead to important insights on the mechanisms and/or treatments of these diseases. Studies providing the most insight into these processes have utilized, as models, the infection of polarized monolayers of MDCK cells by enveloped RNA viruses. Such studies have shown that certain viruses such as influenza virus and Sendai virus bud only from the apical domain, whereas other viruses such as vesicular stomatitis virus bud exclusively from the basolateral domain. This project proposes to clone, express and follow the transport in eukaryotic cells of proteins encoded in the viral genome of one of these viruses, Sendai virus, and its variant, Fl-R. The two viruses differ in their site of budding in polarized cells. In other viral systems, the envelope glycoproteins, when expressed without any other viral genes, have been shown to localize in the same domain from which the virus ultimately buds. This indicates that their polypeptide backbones contain a signal for polarized transport. We will first test to see if this holds true for the Sendai viral proteins. Assuming it does, we hope to utilize their difference in transport site to identify sorting signals in the membrane proteins. The differences in the RNA and protein sequences of the two viruses have recently been published. Utilizing this information we will use site specific mutagenesis to systematically change the variant sequences back to wild-type. Each possible combination of the wild-type and variant sequence will be expressed and the transport followed. Thus, we should be able to identify the mutation(s) responsible for the difference in polarized expression of the proteins.